Method For Producing Fractions of a Milk Composition

ABSTRACT

The invention relates to a method for producing fractions of a milk composition by treating a milk composition with a phospholipase and separating it into at least two fractions with different fat content. The fractions obtained have improved properties for producing food products. The invention further relates to a method for producing food products from one or more of the fractions obtained. In further aspects the invention relates to methods for producing condensed milk, milk powder, butter and dairy spread from a milk composition treated with a phospholipase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/720,651 filed on Jun. 1, 2007 (pending), which is a 35 U.S.C. 371national application of PCT/DK2005/000810 filed Dec. 21, 2005, whichclaims priority or the benefit under 35 U.S.C. 119 of Danish applicationnos. PA 2004 01972 filed Dec. 21, 2004 and PA 2005 00102 filed Jan. 20,2005 and U.S. provisional application nos. 60/638,279 filed Dec. 21,2004 and 60/645,780 filed January 21, 2005, the contents of which arefully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for producing fractions of amilk composition treated with phospholipase and to a method forproducing a food product from one or more of the fractions, as well asfood products obtainable by the method. Furthermore the inventionrelates to methods for producing condensed milk, milk powder, butter anddairy spread.

BACKGROUND OF THE INVENTION

When milk is processed into dairy or other food products there are anumber of properties of the milk that are desirous, depending on theproduct to be produced, e.g. low tendency to produce fouling ofequipment, good emulsification properties and high heat stability.Specifically, for production of butter a high yield of butter from thecream is desirous, and when producing liquid acidified milk drinks, alow viscosity may be desired.

Milk used for production of dairy products is very often heat treated,e.g. by pasteurisation. A well-known problem when heat treating milk isfouling of the heating surfaces, i.e. that milk solids are deposited onthe surfaces decreasing the heat transfer rate. This problem is knowne.g. with plate heat exchangers and in evaporators. Fouling is also animportant problem in relation to a number of other dairy processes, e.g.membrane filtration. Fouling is related to the wetting of the, usuallyhydrophilic, surfaces of processing equipment, and the wettingproperties are largely determined by surface tension. According toParamalingam et al.: On the fouling of falling film evaporators due tofilm break-up (2000), Food and Bioproducts Processing, 78, pp. 79-84,surface tension is an important factor in fouling of falling filmevaporators. Furthermore, it has been shown that the higher the contactangle, the less material is deposited on heating surfaces during heattreatment of milk (0. Santos et al. (2004): Effect of surface and bulksolution properties on the adsorption of whey protein onto steelsurfaces at high temperature.

In: Santos, 0.: Whey protein adsorption and aggregation on modifiedstainless steel surfaces in relation to fouling, Doctoral thesis, LundUniversity, Lund, Sweden). The contact angle is increased when thesurface tension of milk is decreased. Fouling decreases efficiency ofprocessing equipment and increases the need for cleaning of theequipment, leading to lost production time. There is a desire todecrease the rate of fouling of dairy process equipment.

Fouling is often caused by deposition of milk proteins, e.g.beta-lactoglobulin, on solid surfaces, and may be reduced by replacingmilk proteins with other substances at the liquid/solid interface.

Milk comprises phospholipids. The phospholipids are associated with themilk fat due to its non-polar, lipophilic properties. Phospholipids maybe hydrolysed by phospholipase into lysophospholipid, which may in turnbe hydrolysed by a lysophospholipase (phospholipase type B). WO 00/54601(Novozymes A/S) discloses a method for producing cheese, comprisingtreating the cheese milk with a phospholipase.

SUMMARY OF THE INVENTION

The inventors have found that when treating a milk composition with aphospholipase, and separating the milk composition into two or morefractions with different fat content, it is possible to producefractions with novel and advantageous properties for the production offood products, e.g. fractions with reduced surface tension, fractionsthat lead to increased yield of butter upon churning, and/or fractionsthat lead to improved properties of milk powder products produced therefrom.

Consequently, the invention relates to a method for producing fractionsof a milk composition, comprising: i) treating a milk composition with aphospholipase; and ii) separating the treated milk composition into atleast two fractions with different fat content. In further aspects theinvention relates to the fractions of a milk composition obtainable bythe method of the invention, methods for producing food products, andfood products obtainable by using methods of the invention. In a furtheraspect the invention relates to methods for producing condensed milk,milk powder, butter and dairy spread, and condensed milk, milk powder,butter and dairy spread obtainable by the method.

DETAILED DISCLOSURE OF THE INVENTION

Milk Composition

Milk may be the lacteal secretion of any mammal, e.g., cow, sheep, goat,buffalo or camel. A milk composition according to the invention may beany liquid composition comprising milk fat.

A milk composition may comprise one or more milk fractions. In oneembodiment of the invention a milk composition is a milk fraction. Amilk fraction may be any fraction of milk such as e.g. skim milk, buttermilk, whey, cream, milk powder, whole milk powder, skim milk powder. Inanother embodiment of the invention the milk composition comprises milk,skim milk, butter milk, whole milk, whey, cream, or any combinationthereof. In a further embodiment the milk composition consists of milk.The content of milk fat in the milk composition may be controlled toachieve the desired effect, e.g. the desired amount of milk fat andphospholipase hydrolysis products in the fractions obtained whensubjecting the milk composition to the method of the invention.

In further embodiments of the invention, the milk composition isprepared, totally or in part, from dried milk fractions, such as, e.g.,whole milk powder, skim milk powder, casein, caseinate, total milkprotein, butter, anhydrous milk fat, or buttermilk powder, or anycombination thereof.

The milk composition may be standardised to the desired composition byremoval of all or a portion of any of the milk components and/or byadding thereto additional amounts of such components. This may be donee.g. by separation of milk into cream and skim milk at arrival to thedairy. Thus, the milk composition may be prepared as done conventionallyby fractionating milk and recombining the fractions so as to obtain thedesired final composition of the milk composition. The separation may bemade in continuous centrifuges leading to a skim milk fraction with verylow fat content (i.e. e.g. <0.5%) and cream with e.g. >35% fat. The milkcomposition may be prepared by mixing cream and skim milk in appropriateamounts to achieve the desired fat content. In another embodiment theprotein and/or casein content is standardised by the use of UltraFiltration.

In one embodiment of the invention the milk composition is raw milk,i.e. milk which has not been subjected to a separation process or to aheat treatment. In another embodiment the milk composition is milk thathave been heat treated, e.g. by pasteurisation or thermalisation.

The Enzymatic Treatment:

The enzymatic treatment in the process of the invention may be conductedby dispersing the phospholipase into the milk composition and allowingthe enzyme reaction to take place at an appropriate holding-time at anappropriate temperature. The treatment with phospholipase may be carriedout at conditions chosen to suit the selected phospholipase according toprinciples well known in the art. The treatment may also be conducted bycontacting the milk composition with phospholipase that has beenimmobilised.

The phospholipase treatment may be conducted at any suitable pH, such ase.g., in the range 2-10, such as, at a pH of 4-9 or 5-7. It may bepreferred to let the phospholipase act at the natural pH of the milkcomposition.

The phospholipase treatment may be conducted at any appropriatetemperature, e.g. in the range 1-70° C., such as 2-60° C. Thephospholipase treatment may be conducted at a low temperature, e.g. inthe storage tank at the farmer or at the dairy, e.g. at a temperature of2-7° C.

In one embodiment of the invention phospholipase is added to milk at thefarm, and the phospholipase is allowed to react in the milk at lowtemperature during storage at the farm, and/or during transportation tothe dairy plant, and optionally during storage at the dairy plant. Inanother embodiment phospholipase is added to the milk at reception atthe dairy plant and allowed to react in the milk during storage at thedairy plant.

In another embodiment of the invention the phospholipase is allowed toreact in the milk composition at the temperature of separation. If themilk is separated in a conventional continuous centrifuge the separationtemperature will usually be in the range 40-70° C.

Optionally, after the phospholipase has been allowed to act on the milkcomposition, the phospholipase enzyme protein may be removed, reduced,and/or inactivated.

The phospholipase is added in a suitable amount to achieve the desireddegree of hydrolysis under the chosen reaction conditions. A suitabledosage of a phospholipase A may e.g. be in the range 0.003-0.3 mg enzymeprotein per g milk fat, preferably 0.01-0.3 mg enzyme protein per g milkfat, more preferably, 0.03 mg enzyme protein per g milk fat. A suitabledosage of a phospholipase B may e.g. be in the range 0.005-0.5 mg enzymeprotein per g milk fat, preferably 0.01-0.5 mg enzyme protein per g milkfat, more preferably 0.05 mg enzyme protein per g milk fat.

In cow milk, the lecithin constitutes normally more than 95% of thephospholipids in milk whereas the lysolecithin is approximately 1% ofthe phospholipids. The amount of phosphatidyl choline (PC) andphosphatidyl ethanolamine (PE) is approximately 40% of the lecithin. Inone embodiment of the invention, the lecithin content of the milkcomposition is reduced by at least 5%, such as at least 10%, at least15%, at least 20%, or at least 25%, as a result of the phospholipasetreatment. In another embodiment the amount of phosphatidyl cholineand/or phosphatidyl ethanolamine in the milk composition is reduced byat least 20%, such as at least 40%, at least 60%, or at least 70% as aresult of the phospholipase treatment.

Separation into at Least Two Fractions

According to the present invention a milk composition is separated intoat least two fractions with different fat content. The separation may beperformed by any method known in the art for separating milkcompositions. In one embodiment milk is separated by centrifugation,e.g. in a conventional continuous centrifuge. In another embodiment milkis separated by filtration, e.g. by micro- or ultrafiltration. In allembodiments the properties of the separation technique and equipmentmust be such that the ratio between milk fat and phospholipid hydrolysisproducts in at least two of the fractions produced is different from theratio in the phospholipase treated milk composition before separation.In one embodiment of the invention milk is separated into two fractions,e.g. into cream and skim milk. The fat content of skim milk will usuallybe below 0.6% (weight/weight) such as below 0.2%, the fat content ofcream will usually be in the range 30-45% (weight/weight), such as inthe range 35-40%. In one embodiment of the invention the fat content ofone of the fractions obtained is at least the double of the fat contentof another fraction.

In one embodiment of the invention a milk composition treated with aphospholipase is separated into at least two liquid fractions in stepii) of the method of the invention. A liquid milk composition or milkfraction is a milk composition or milk fraction that flows freely underthe processing conditions used for the separation into milk fractions,i.e. a milk composition or milk fraction wherein substantially all ofthe milk casein is in a dispersed un-coagulated state. Examples ofliquid milk compositions/milk fractions are milk, skim milk, cream, andpermeate and retentate from ultra- and microfiltration, as opposed toe.g. cheese and rennet coagulated milk which are non-liquid.

In one embodiment the invention relates to fractions obtainable by themethod of the invention. In a further embodiment the invention relatesto fractions obtainable by the invention having a different fat contentthan the original milk composition that is treated with phospholipase instep i).

Properties of Fractions

The surface tension of the fractions may be affected by the method ofthe invention. In one embodiment of the invention the surface tension ofat least one of the fractions is reduced compared to a similar fractionproduced from a milk composition that has not been treated withphospholipase. In another embodiment the surface tension of at least onefraction is reduced or increased compared to a similar fraction producedfrom a milk composition that has not been treated with a phospholipase,but which fraction has been treated with phospholipase after theseparation step. In a further embodiment the content of phospholipidhydrolysis products is higher or lower than in a similar fractionproduced from a milk composition that has not been treated with aphospholipase, but which fraction has been treated with phospholipaseafter the separation step. In a still further embodiment the content oflysolecithin is higher or lower than in a similar fraction produced froma milk composition that has not been treated with a phospholipase, butwhich fraction has been treated with a phospholipase after theseparation step.

The method of the invention affects the properties of at least one ofthe fractions obtained compared to a similar fraction from milk that hasnot been treated with a phospholipase. In one embodiment of theinvention the properties of at least one of the fractions obtained aredifferent from the properties of a similar fraction produced from a milkcomposition that has not been treated with a phospholipase, but whichfraction has been treated with phospholipase after the separation step.The property affected may e.g. be the fouling observed when the fractionis subjected to a heat treatment, e.g. pasteurisation in a plate heatexchanger or Ultra High Temperature (UHT) treatment by steam injection;evaporation, e.g. in a falling film evaporator; or a membrane separationprocess, e.g. ultrafiltration or microfiltration. The method of theinvention may reduce the degree of fouling observed with at least one ofthe fractions produced. The degree of fouling may be determined as theamount of solid material absorbed e.g. to the plates of a plate heatexchanger, to the walls of a falling film evaporator, or to the membraneof a membrane filtration unit. Fouling of surfaces may be observed in anexperimental setup by ellipsometry, e.g. as described in O. Santos etal. (2004): Effect of surface and bulk solution properties on theadsorption of whey protein onto steel surfaces at high temperature. In:Santos, O.: Whey protein adsorption and aggregation on modifiedstainless steel surfaces in relation to fouling, Doctoral thesis, LundUniversity, Lund, Sweden.

The method of the invention may also affect the churnability of at leastone of the fractions obtained, e.g. cream obtained by the method of theinvention may need less time and/or less vigorous mechanical treatmentto form butter and/or the yield of butter obtained may be higher,compared to cream obtained from a milk composition that has not beentreated with a phospholipase, or cream that has been treated with aphospholipase without subsequent separation. Likewise, theemulsification properties may be improved, e.g. the emulsion stabilityor emulsion capacity. Additionally, the heat stability of one or more ofthe fractions may be improved, e.g. the temperature and/or time neededto induce flocculation or coagulation may be increased. Properties offood products produced from one or more of the fractions obtained by themethod of the invention may be affected compared to properties of foodproducts produced from a similar fraction produced from a milkcomposition that has not been treated with a phospholipase, but whichfraction has been treated with phospholipase after the separation step.E.g. the appearance and texture, e.g. viscosity, of acidified dairyproducts; emulsification properties of milk; and the solubility andwettability of milk powders produced from the milk; may all be affected.

Food Product

In one embodiment the invention relates to a method for producing a foodproduct from one or more fractions obtained by the method of theinvention. A food product according to the invention may be any foodproduct wherein milk or one or more milk fractions may be used as aningredient, e.g. a meat product, a fish product, bread, cake, cookies,biscuits, confectionary products such as e.g. chocolate, dressings,sauces, and condiments. A food product may be produced by any methodknown in the art, and milk fractions obtained by the method of theinvention may be used in the same way as conventional milk or milkfractions are normally used in production of food products. The milkfraction or fractions to be used in the production of a food product maybe subjected to any processing known in the art to be useful in theproduction of a food product. One or more milk fractions may e.g. beheat treated, e.g. in a plate heat exchanger or in an evaporator orsubjected to ultra- or microfiltration.

In one embodiment of the invention a food product is a dairy product.One or more milk fractions obtained by the method of the invention maybe used for production of any dairy product, such as e.g. milk, e.g.skim milk, partly skimmed milk, or cream, for direct consumption;acidified milk, e.g. yoghurt; ice cream; milk powder, e.g. skim milkpowder, whole milk powder, or butter milk powder; butter; ghee;anhydrous milk fat; buttermilk; condensed milk; sweetened condensedmilk; dulche de leche; coffee whitener; coffee creamer; or cheese.Cheese may be any kind of cheese, e.g. fresh cheese, hard cheese, creamcheese, white mould cheese, blue mould cheese, process cheese, or pastafilata type cheese. A dairy product according to the invention may alsobe a recombined dairy product produced from dried milk components andoptionally anhydrous milk fat, e.g. liquid whole milk or butter madefrom skim milk powder and anhydrous milk fat.

A food product of the invention may often be produced from two or morefractions of the invention, e.g. from a combined milk fraction. In thisway a combined milk fraction with the desired composition and fatcontent may be prepared, and such a combined milk fraction may have adifferent content of phospholipid hydrolysis product than a similarcombined milk fraction that is treated with phospholipase afterseparation into fractions and subsequent mixing into a combinedfraction. By mixing fractions of the invention into combined milkfractions it is thus possible to obtain combined milk fractions with arange of compositions and with novel and improved properties.

In one embodiment of the invention a combined milk fraction is producedby mixing two or more milk fractions with differing fat content. Acombined milk fraction may preferably have a different fat content thanthe milk composition from which the milk fractions were obtained. Bymixing milk fractions in a desired ratio into a combined milk fraction,a combined milk fraction with a desired fat content may be obtained. Inone embodiment of the invention a combined milk fraction is obtained bymixing two or more milk fractions of the invention with differing fatcontent in a ratio resulting in a combined milk fraction with adifferent fat content than the milk composition from which the fractionswere obtained. A combined milk fraction may e.g. be produced by mixingskim milk and cream produced from phospholipase treated milk in desiredamounts. In one embodiment of the invention a combined milk fractioncomprises at least 0.5% (weight/weight) fat, such as at least 1% fat, orat least 1.5% fat. A combined milk fraction may be used for theproduction of a food product of the invention, e.g. a dairy product.

In a further embodiment of the invention a milk fraction obtained by themethod of the invention is combined with a milk fraction obtained from amilk composition that has not been treated with a phospholipase toproduce a combined milk fraction. In a still further embodiment of theinvention a milk fraction is obtained from a milk composition that hasnot been treated with a phospholipase and the obtained milk fraction isthen treated with a phospholipase to hydrolyse phospholipids, and thetreated milk fraction is mixed with a milk fraction obtained by themethod of the invention. Thus, in one embodiment the invention relatesto: A method for producing a combined milk fraction, comprising: a)treating a first milk composition with a phospholipase; and b)separating the treated milk composition into at least two milkfractions; and c) separating a second milk composition into at least twofractions, and d) treating at least one milk fraction obtained from saidsecond milk composition as a result of step c with a phospholipase; ande) combining at least one milk fraction obtained from said first milkcomposition as a result of step b) with at least one phospholipasetreated milk fraction obtained as a result of step d). In a furtherembodiment cream is obtained from milk that has not been treated with aphospholipase, this cream is then treated with a phospholipase, and thephospholipase treated cream is mixed with skim milk obtained by themethod of the invention. In this way a combined milk fraction can beproduced with an increased level of lysophopsholipids, sincephospholipids associated with the milk fat in the cream obtained fromuntreated milk is combined with lysophopsholipids present in the skimmilk phase of milk that has been treated with a phospholipase beforeseparation. The invention also relates to food products obtained orobtainable by the methods of the invention.

Method for Producing Condensed Milk or Milk Powder

In one aspect the invention relates to a method for producing condensedmilk or milk powder by treating a milk composition with a phospholipaseand producing condensed milk or milk powder from the treated milkcomposition. Production of condensed milk and milk powder involves theremoval of water by evaporation. Treatment of a milk composition withphospholipase may reduce the surface tension, and a reduced surfacetension may reduce the degree of fouling observed e.g. duringevaporation, which improves the economy of the process. Treatment of amilk composition with a phospholipase may be performed by any suitablemethod, e.g. as described elsewhere in this document, or as described inWO 00/54601 in relation to production of cheese. The phospholipasetreatment may e.g. be performed by dispersing the phospholipase in themilk composition. The phospholipase treated milk composition may be heattreated to inactivate the phospholipase before the evaporating or dryingstep.

Condensed milk according to the invention may be produced from aphospholipase treated milk composition by any suitable method known inthe art. Usually condensed milk is produced from milk that isstandardised to the desired fat content and subjected to heat treatment,e.g. at 100-120° C. for 1-3 minutes. The milk is then usually subjectedto evaporation, e.g in a falling film evaporator where water is removedunder reduced pressure, e.g. at a temperature of 65-70° C., until thedesired solids content has been achieved. The condensed milk may behomogenised to disperse the fat globules and prevent coagulation if thecondensed milk is to be sterilised, homogenisation may e.g. be performedin a two-stage homogeniser at a pressure of e.g. 12-25 MPa. Thecondensed milk may be filled onto cans or any other suitable containers,and will usually be subjected to sterilisation, e.g. at 110-120° C. for15-20 minutes. Alternatively the condensed milk may be subjected to UHTtreatment before filling onto containers, e.g. at 140° C. for 3 seconds.For production of sweetened condensed milk sugar may be added at anappropriate step during the process, e.g. dry sugar may be added beforeheat treatment of the milk, or sugar syrup may be added duringevaporation. Sugar will usually be added in an amount resulting in atleast 62.5% sugar in the aqueous phase of the finished product. In orderto control the crystallisation of lactose in sweetened condensed milk,it may be cooled rapidly under agitation after evaporation, and lactosecrystals may be added as dry crystals or as a slurry when the milk hascooled to crystallisation temperature, usually around 30° C. Sweetenedcondensed milk does not need sterilisation because of the osmoticpressure caused by the added sugar.

Milk powders are usually produced from heat treated milk; additionallythe milk may be subjected to e.g. bactofugation or microfiltration inorder to decrease the bacterial count of the milk. Milk powder may beproduced by roller drying of milk, wherein the milk is distributed in athin layer on the surface of heated drums whereby water is evaporated.The dried layer may be scraped of by knifes and may be ground intoflakes and/or grinded into powder. Alternatively, milk powder may e.g.be produced by spray drying. Before spray drying the milk is usuallyconcentrated by evaporation to a dry matter content of e.g. 45-55%, e.g.in a falling film evaporator. The concentrated milk may them be pumpedto a drying tower, wherein it is dispersed into very fine droplets whichare mixed into a stream of hot air whereby the water is quicklyevaporated. The dried milk particles are then separated from the hotair, e.g. in a cyclone. The process may also be performed as a two-stageprocess wherein the milk particles from the drying tower is subjected tofurther drying in a fluid bed dryer. Furthermore the process may beconducted as a three-stage process with two fluid bed drying steps,where the first fluid bed drying may be integrated in the drying tower.The drying equipment may be designed to produce a powder with theability to be very quickly redissolved (“instant powder”). This effectmay e.g. be achieved by rehumidifying the dried particles, either in thedrying tower or in the fluid bed dryer, causing the particles to adhereand form agglomerates. Milk powder may be produced e.g. from skim milk,whole milk or butter milk. In one embodiment of the invention milkpowder is skim milk powder. Milk powder produced by the method of theinvention may e.g. be used in dairy plants that produce recombinantdairy products. Other examples of the utilisation of milk powders aree.g. in production of coffee whiteners and coffee creamers. The heatstability of the milk composition used for production of milk powdersand of the milk powder is an important quality parameter. In oneembodiment of the invention the heat stability of powders produced bythe method of the invention is increased compared to milk powderproduced by a conventional method.

The invention also relates to condensed milk and milk powder obtainableby the methods of the invention.

Method for Producing Butter or Dairy Spread

In one aspect the invention relates to a method for producing butter ordairy spread by treating a milk composition with a phospholipase andproducing butter or dairy spread from the treated milk composition.

Local regulations may differ with regard to the definitions of butter,dairy spread and similar products. In the present context, butter isunderstood as a water-in-oil emulsion suitable for human consumption,wherein the fat phase is comprised of milk fat, and which additionallycomprises non-fat milk solids, and is produced from cream. The fatcontent of butter is usually between 80% (weight/weight) and 95%, butmay depend on local regulations, e.g. light butter or reduced fat buttermay be produced e.g. with a fat content between 40% and 80% or evenbelow 40%. Butter may be produced from sweet (non-acidified) cream orsour (acidified) cream. Butter may additionally comprise salt, and whereregulations allow further additives such as e.g. emulsifiers andflavours. A dairy spread in the present context is understood as awater-in-oil emulsion suitable for human consumption, wherein the fatphase comprises milk fat and non-milk fat, and which may compriseadditional milk and/or non-milk constituents. The fat content of a dairyspread usually varies between 35% (weight/weight) and 95%, but may evenbe below 35% for low fat dairy spreads. The non-milk fat component isusually vegetable fat, such as e.g. soybean oil or rapeseed oil. Theratio of milk fat to non-milk fat may be any ratio suitable to achievethe desired effect. If the desired effect is a butter-like product whichis ready spreadable at refrigerator temperature usually between 15% and40% of the fat is non-milk fat such as vegetable oil. A dairy spreadmay, depending on local regulations, comprise additional components suchas e.g. salt, emulsifiers, stabilisers, and flavours.

Treatment of a milk composition with a phospholipase may be performed byany suitable method, e.g. as described elsewhere in this document, or asdescribed in WO 00/54601 in relation to production of cheese. Thephospholipase treatment may e.g. be performed by dispersing thephospholipase in the milk composition. In one embodiment of theinvention cream is treated with a phospholipase before it is used forproduction of butter or dairy spread.

The phospholipase treated milk composition may be heat treated toinactivate the phospholipase. In one embodiment of the invention a milkcomposition to be used for production of dairy spread is mixed with acomposition comprising non-milk fat before treatment with aphospholipase.

Butter or dairy spread according to the invention may be produced bysubjecting a phospholipase treated milk composition, e.g. cream, to anyprocess known in the art suitable for producing butter or dairy spread.Cream to be used for production of butter or dairy spread is usuallypasteurised to destroy unwanted microorganisms and/or enzymes, and maybe acidified, e.g. by fermenting the cream with lactic acid bacteria.

Butter may e.g. be produced in a churn. A churn for butter productionmay e.g. be a cylindrical, conical, cubical or tetrahedral tank whichcan be rotated to agitate the cream to press fat out of milk fatglobules to form free fat that coalesce into butter grains. Theremaining liquid, the buttermilk, may be drained of and usually thebutter is worked further in the churn to expel additional liquid and todistribute the remaining liquid evenly throughout the butter. The airpressure in the churn may be reduced during working to reduce the amountof air captured in the butter. Butter may also be produced in acontinuous buttermaking machine. In a continuous buttermaking machinecream is transported continuously through several sections, e.g. achurning section, a separation section, a squeeze drying section and aworking section. In the buttermaking machine the cream is churned toform butter grains, butter milk is separated off, and the butter isworked to distribute the liquid and expel air. If sweet cream is used asthe raw material, lactic acid and/or a lactic bacteria starter culturemay be mixed into the butter to impart acidity and flavour to thebutter.

A dairy spread may e.g. be produced in a similar process as butter. Inthat case cream and non-milk fat, and additional ingredients, may bemixed before feeding to the butter production process.

Butter or dairy spreads may also be produced from cream concentrated toa milk fat content of e.g. 75-82% in a cream concentrator centrifuge.The concentrated cream may be mixed with e.g. water, salt and otheradditives and, if a dairy spread is to be produced, with non-milk fat,e.g. vegetable oil. The mixture may then be fed to scraped surfacecoolers where the emulsion is inversed to create a water-in-oilemulsion. The inverted emulsion may further be fed to pin kneadingmachines to be worked.

Enzymes to be Used in the Processes of the Invention:

Phospholipids, such as lecithin or phosphatidylcholine, consist ofglycerol esterified with two fatty acids in an outer (sn-1) and themiddle (sn-2) positions and esterified with phosphoric acid in the thirdposition; the phosphoric acid, in turn, may be esterified to anamino-alcohol. Phospholipases are enzymes which participate in thehydrolysis of phospholipids. Several types of phospholipase activity canbe distinguished, including phospholipase A, which can further beclassified as phospholipase A₁ (EC 3.1.1.32) or A₂ (EC 3.1.1.4.), whichhydrolyze one fatty acyl group (in the sn-1 and sn-2 position,respectively) to form lysophospholipid. Phospholipase B (EC 3.1.1.5)hydrolyzes the remaining fatty acyl group in lysophospholipid. Otherphospholipases are phospholipase C (EC 3.1.4.3) and phospholipase D (EC3.1.4.4).

A phospholipase to be used in the method of the invention may be anyphospholipase or any combination of phospholipases. In one embodimentthe phospholipase to be used in the invention is a phospholipase Aand/or a phospholipase B. The phospholipase activity may be provided byenzymes having other activities as well, such as e.g. a lipase withphospholipase activity. The phospholipase activity may e.g. be from alipase with phospholipase side activity. In other embodiments of theinvention the phospholipase enzyme activity is provided by an enzymehaving essentially only phospholipase activity and wherein thephospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, e.g. of animal origin (such as,e.g. mammalian), e.g. from pancreas (e.g. bovine or porcine pancreas),or snake venom or bee venom. Alternatively, the phospholipase may be ofmicrobial origin, e.g. from filamentous fungi, yeast or bacteria, suchas the genus or species Aspergillus, e.g. A. niger; Dictyostelium, e.g.D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus;Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g.R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S.libertiana; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W.sclerotiorum; Bacillus, e.g. B. megaterium, B. subtilis; Citrobacter,e.g. C. freundii; Enterobacter, e.g. E. aerogenes, E. cloacaeEdwardsiella, E. tarda; Erwinia, e.g. E. herbicola; Escherichia, e.g. E.coli; Klebsiella, e.g. K. pneumoniae; Proteus, e.g. P. vulgaris;Providencia, e.g. P. stuartii; Salmonella, e.g. S. typhimurium;Serratia, e.g. S. liquefasciens, S. marcescens; Shigella, e.g. S.flexneri; Streptomyces, e.g. S. violeceoruber; Yersinia, e.g. Y.enterocolitica. Thus, the phospholipase may be fungal, e.g. from theclass Pyrenomycetes, such as the genus Fusarium, such as a strain of F.culmorum, F. heterosporum, F. solani, F. venenatum, or a strain of F.oxysporum. The phospholipase may also be from a filamentous fungusstrain within the genus Aspergillus, such as a strain of Aspergillusawamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nigeror Aspergillus oryzae. A preferred phospholipase is derived from astrain of Fusarium, particularly F. oxysporum, e.g. from strain DSM 2672as described in WO 98/26057, especially described in claim 36 and SEQ IDNO. 2 of WO 98/26057. In further embodiments, the phospholipase is aphospholipase as disclosed in WO 00/32758 (Novozymes A/S, Denmark).

Enzyme Sources and Formulation

The phospholipase used in the process of the invention may be derived orobtainable from any of the sources mentioned herein. The term “derived”means in this context that the enzyme may have been isolated from anorganism where it is present natively, i.e. the identity of the aminoacid sequence of the enzyme are identical to a native enzyme. The term“derived” also means that the enzymes may have been producedrecombinantly in a host organism, the recombinant produced enzyme havingeither an identity identical to a native enzyme or having it a modifiedamino acid sequence, e.g. having one or more amino acids which aredeleted, inserted and/or substituted, i.e. a recombinantly producedenzyme which is a mutant and/or a fragment of a native amino acidsequence. Within the meaning of a native enzyme are included naturalvariants. Furthermore, the term “derived” includes enzymes producedsynthetically by e.g. peptide synthesis. The term “derived” alsoencompasses enzymes which have been modified e.g. by glycosylation,phosphorylation etc., whether in vivo or in vitro. The term “obtainable”in this context means that the enzyme has an amino acid sequenceidentical to a native enzyme. The term encompasses an enzyme that hasbeen isolated from an organism where it is present natively, or one inwhich it has been expressed recombinantly in the same type of organismor another, or enzymes produced synthetically by e.g. peptide synthesis.With respect to recombinantly produced enzyme the terms “obtainable” and“derived” refers to the identity of the enzyme and not the identity ofthe host organism in which it is produced recombinantly.

Accordingly, the phospholipase may be obtained from a microorganism byuse of any suitable technique. For instance, a phospholipase enzymepreparation may be obtained by fermentation of a suitable microorganismand subsequent isolation of a phospholipase from the resulting fermentedbroth or microorganism by methods known in the art. The phospholipasemay also be obtained by use of recombinant DNA techniques. Such methodnormally comprises cultivation of a host cell transformed with arecombinant DNA vector comprising a DNA sequence encoding thephospholipase in question and the DNA sequence being operationallylinked with an appropriate expression signal such that it is capable ofexpressing the phospholipase in a culture medium under conditionspermitting the expression of the enzyme and recovering the enzyme fromthe culture. The DNA sequence may also be incorporated into the genomeof the host cell. The DNA sequence may be of genomic, cDNA or syntheticorigin or any combinations of these, and may be isolated or synthesizedin accordance with methods known in the art.

Suitable phospholipases are available commercially. Examples arepancreas-derived phospholipase such as Lecitase® (manufactured byNovozymes A/S, Bagsvaerd, Denmark), and microbially derivedphospholipase such as YieldMax® (Chr. Hansen A/S and Novozymes A/S,Denmark).

In the process of the invention the phospholipase may be purified. Theterm “purified” as used herein covers phospholipase enzyme protein freefrom components from the organism from which it is derived. The term“purified” also covers phospholipase enzyme protein free from componentsfrom the native organism from which it is obtained, this is also termed“essentially pure” phospholipase and may be particularly relevant forphospholipases which are naturally occurring and which have not beenmodified genetically, such as by deletion, substitution or insertion ofone or more amino acid residues.

Accordingly, the phospholipase may be purified, viz. only minor amountsof other proteins being present. The expression “other proteins” relatein particular to other enzymes. The term “purified” as used herein alsorefers to removal of other components, particularly other proteins andmost particularly other enzymes present in the cell of origin of thephospholipase. The phospholipase may be “substantially pure”, i.e. freefrom other components from the organism in which it is produced, i.e.,e.g., a host organism for recombinantly produced phospholipase.Preferably, the enzymes are at least 75% (w/w) pure, more preferably atleast 80%, 85%, 90% or even at least 95% pure. In a still more preferredembodiment the phospholipase is an at least 98% pure enzyme proteinpreparation. In other embodiments the phospholipase is not naturallypresent in milk.

The terms “phospholipase” includes whatever auxiliary compounds that maybe necessary for the catalytic activity of the enzyme, such as, e.g. anappropriate acceptor or cofactor, which may or may not be naturallypresent in the reaction system.

The phospholipase may be in any form suited for the use in question,such as e.g. in the form of a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a protected enzyme.Granulates may be produced, e.g. as disclosed in U.S. Pat. No. 4,106,991and U.S. Pat. No. 4,661,452, and may optionally be coated by methodsknown in the art. Liquid enzyme preparations may, for instance, bestabilized by adding stabilizers such as a sugar, a sugar alcohol oranother polyol, lactic acid or another organic acid according toestablished methods. Protected enzymes may be prepared according to themethod disclosed in EP 238,216.

Phospholipase Activity (LEU)

Phospholipase activity may be determined by the following method:Lecithin is hydrolyzed under constant pH and temperature, and thephospholipase activity (LEU) is determined as the rate of titrant (0.1NNaOH) consumption during neutralization of the liberated fatty acid bycomparison to a known enzyme standard (available from Novozymes A/S,Bagsvaerd, Denmark). The substrate may be soy lecithin(L-alfa-Phosphotidyl-Choline) at a concentration of 20 g/1000 ml, andthe conditions are pH 8.00, 40.0° C., reaction time 2 min.

EXAMPLE 1

Un-homogenized whole milk was used to produce three different samples ofskim milk and two samples of cream by the following methods:

Sample 1.1

The milk was incubated at 40° C. for 30 minutes with a phospholipasefrom Fusarium venenatum. Enzyme dosage was 700 LEU/I milk. The enzymereaction was terminated by heating the milk to 72° C. for 10 minutes.The milk was centrifuged at 3000 g for 20 minutes and separated in skimmilk and cream.

Sample 1.2

The milk was held at 40° C. for 30 minutes without addition ofphospholipase and heat treated at 72° C. for 10 minutes. The milk wascentrifuged at 3000 g for 20 minutes and separated in skim milk andcream.

Sample 1.3

Skim milk from sample 1.2 was incubated at 40° C. for 30 minutes with aphospholipase from Fusarium venenatum. Enzyme dosage was 700 LEU/I milk.The enzyme reaction was terminated by heating the milk to 72° C. for 10minutes

Surface tension of the three skim milk samples using a tensiometer(Sigma 70, KSV Instruments Ltd., Finland) with the Wilhelmy platetechnique. Measurement duration was one hour.

The following results were recorded for the surface tension (mN/m):

Start 15 min 30 min 45 min 60 min Skim milk from sample 1.1 33.5 33.233.1 33.1 32.9 Skim milk from sample 1.2 42.4 41.6 40.5 40.0 39.6 Skimmilk from sample 1.3 35.2 34.6 34.5 34.4 34.3

EXAMPLE 2

The two cream fractions obtained from sample 1.1 and sample 1.2 ofexperiment 1 were used for churning of butter in a Kitchen Aid whippingmachine operated at maximum speed. 250 g cream was cooled to 5° C.before churning and the whipping/churning was performed at roomtemperature.

The butter and buttermilk was separated using a sieve and the amount ofbutter was measured by weighing. The cream separated from the enzymetreated whole milk of sample 1.1 gave a butter yield of 34.76% of thecream, whereas the cream from the untreated milk of sample 1.2 resultedin 33.32%.

EXAMPLE 3

The two skim milk fractions from the sample 1.1 and sample 1.2 ofexample 1 were used for production of acidified milk. 9 parts skim milkwas incubated with 1 part commercial acidified buttermilk and incubatedfor 20 hours at room temperature. The incubation was made directly inthe device used for viscosity measurements (Rapid Visco Analyzer RVA-4.Newport Scientific, Australia). This consists of a measure cylindermounted with a spindle. Five sample of each treatment were prepared. Thespindle was rotated at 100 rpm and the viscosity was recordedcontinuously during 15 minutes of rotation. The average viscosity was362 cp (centipoise) for the enzyme treated acidified skim milk fromsample 1.1 and 406 cp for the acidified skim milk not treated withenzyme from sample 1.2.

EXAMPLE 4

Un-homogenized whole milk was used to produce two different samples ofskim milk by the following methods:

Sample 4.1

The milk was incubated at 40° C. for 30 minutes with a phospholipasefrom Fusarium venenatum. Enzyme dosage was 350 LEU/I milk. The enzymereaction was terminated by heating the milk to 72° C. for 10 minutes.The milk was centrifuged at 3000 g for 20 minutes and separated in skimmilk and cream.

Sample 4.2

The milk was held at 40° C. for 30 minutes without addition ofphospholipase and heat treated at 72° C. for 10 minutes. The milk wascentrifuged at 3000 g for 20 minutes and separated in skim milk andcream.

Content of phospholipids phosphatidyl choline (PC) and phosphatidylethanolamine (PE) in the whole milk samples and skim milk samples from4.1 and 4.2 was analyzed by HPLC.

The following results were recorded for the content of PC and PE (μg):

PC PE 4.1 raw milk - enzyme treated 0.47 Below detection limit 4.1 skimmilk - enzyme treated 0.20 Below detection limit 4.2 raw milk - noenzyme 6.42 6.33 4.2 skim milk - no enzyme 6.05 5.12

EXAMPLE 5 Replacement of Beta-Lactoglobulin from Air-Water Interface byHydrolyzed Milk Phospholipids

Method: Surface Tension and Elasticity in Dilation

The surface dilatational elasticity and surface tension were determinedby the method of Kokelaar et al. (Kokelaar, J. J., Prins, A. and De Gee,M., J. Colloid Interface Sci., 1991, 146, 507). The technique involves aperiodical expansion and compression of the surface resulting fromraising and lowering of a 10 cm diameter ground glass ring into a vesselcontaining 200-250 ml of sample solution. The change in surface tensionresulting from the induced surface area change was used to calculate thesurface dilatational modulus [E] (surface elasticity). The change inarea was kept at maximum 5% in order to prevent disruption of thesurface layer.

beta-lactoglobulin solution was poured into the sample vessel and themeasurement was started immediately after dipping the grounded glassWilhelmy plate into the sample.

Adsorption of protein to the air-water interface was monitored byfollowing the decrease of surface tension and increase of surfaceelasticity. After 45 minutes, when the surface elasticity was close to aplateau, and 4 ml of phospholipid sample was injected to the solutionunder the liquid surface, outside the ring. The development of surfacetension and [E] were monitored for 155 minutes after the addition. Twoor three replicas of each phospholipid preparation were measured.

Sample Preparation

The Used Milk Phospholipid Preparations Were:

1) purified native milk phospholipid mixture (MPLC),

2) MPLC hydrolyzed by a phospholipase (YieldMax®, Chr. Hansen A/S andNovozymes A/S, Denmark; 60 LEU/mg phospholipid)

3) milk phospholipid concentrate hydrolyzed by pancreatic phospholipase(Lecitase 10L, Novozymes A/S, Bagsvaerd, Denmark) and subsequentlypurified by preparative chromatography.

The start material in all cases was a commercial milk phospholipidconcentrate PSNU18200, from Arla Foods, Nr Vium, Denmark. Thephospholipid composition of the preparations is presented in Table 1.

The samples were dispersed in 20 mM imidazol, 6.5 mM CaCl₂, pH 7.0 andsonicated by a probe sonicator in order to achieve a fine dispersion.

Beta-lactoglobulin genetic variant B was purified as described byKristiansen et al. (Kristiansen, K. R, Otte, J., Ipsen, R. and Qvist,K., Int Dairy J., 1998, 8, 113-118). A stock solution of 2 mg/mlbeta-lactoglobulin was prepared in 20 mM imidazol, 6.5 mM CaCl₂, pH 7.0.The precise concentration was calculated based on absorbance at 280 nm,using an absorptivity of 0.96 mlmg⁻¹cm⁻¹. The stock was diluted in thesame buffer to 10 micromolar concentration immediately before startingthe measurement.

TABLE 1 Phospholipid content (μg/g) in the bulk subphase duringreplacement experiment. Sample PC PE LPC LPE SPH Total Native MPLC 8.69.0 0 0 14.3 32   Hydrolyzed MPLC 1.7* 1.8* 4.6* 4.8* 14.3 27*/32**Purified hydrolysate <1.5 0 4.0 5.9 8.1 18.5 *Estimated based onexpected 80% hydrolysis of PC and PE. **Content before hydrolysis: Onweight basis, the amount is reduced during hydrolysis as fatty acid isreleased. PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; LPC:Lyso-phosphatidylcholine; LPE: Lyso-phosphatidylethanolamine; SPH:Sphingomyelin

Results

Injection of hydrolyzed MPLC or purified hydrolysate in the bulk underthe beta-lactoglobulin surface film caused a sudden drop of surfaceelasticity [E] from approx. 90 mN/m to <50 mN/m. The abolishment ofsurface elasticity indicates rupture of the beta-lactoglobulin surfacefilm and removal of protein from the surface. Simultaneously, thesurface tension was decreased to lower values than those typical forbeta-lactoglobulin. The results show that beta-lactoglobulin wasreplaced by more surface active compounds. Native milk phospholipidmixture did not cause replacement of protein from the air-waterinterface (surface elasticity and surface tension did not changesubstantially upon addition), so clearly the hydrolysis products areessential for the removal of the protein layer.

The results show the ability of hydrolysis products of milkphospholipids to replace whey protein at interfaces.

EXAMPLE 6 Butter from Phospholipase Treated Cream

Cream (38% fat) was heated to 19° C. and treated with a phospholipase(YieldMax®, Chr. Hansen A/S and Novozymes A/S, Denmark; Dosage 1750LEU/I cream) for 2 hours. After enzyme treatment 100 g cream was whippedin a whipping/churning equipment (Slagsahne Geprüfgerät, JAN2Labortecnik, D-2427 Malente/Holst, Germany). Control samples of creamnot treated with phospholipase were whipped using the same procedure.Whipping was continued for 30 seconds after churning occurred (measuredas maximum energy consumption). The amount of butter produced wasweighed after dripping off the buttermilk. Butter yield of controlsamples was 50.30% (average of 4 samples tested), and butter yield ofenzyme treated samples was 54.18%.

Surface tension of cream after enzyme treatment was 31.6 mN/m comparedto control sample 33.8 mN/m.

EXAMPLE 7 Milk Powder from Phospholipase Treated Milk

100 l milk (3.5% fat) was heated in a tank to 40° C. and phospholipasewas added at dosage 175 LEU/l milk (1.84 g enzyme/100 l milk). Thephospholipase was from Fusarium venenatum with activity of 9500 LEU/g.

After 30 minutes treatment the milk was flash pasteurized to inactivatethe enzyme at 80° C.

The milk was evaporated to 40% dry solids in a one-step falling filmevaporator, and spray dried with inlet air temperature of 180° C. andoutlet air temperature of 82° C.

A control sample batch was treated the same way only omitting the enzymeaddition.

Milk powder samples were analysed for solubility index, free fat, andsurface tension. Solubility index was measured as the amount of sedimentin ml after two centrifugations of 13 g reconstituted milk powder in 100ml water at room temperature. The milk powder was added water and mixedin a blender with 3500 rpm. Of the solution 50 ml was centrifuged in 5minutes, the supernatant removed and the sediment suspended in 25 mlwater, and centrifuged. The amount (ml) after the second centrifugationwas given as solubility index.

Free fat is the amount of fat that was extracted by CCl₄ in percentageof total fat content.

Surface tension is measured with the Wilhelmy plate method using atensiometer. (Kruss Tensiometer K10, Kruss GmbH, Hamburg, Germany)

The following results were found:

Control sample Enzyme treated Solubility index (ml) 0.3 0.1 Free fat (%of total fat) 3.52 2.53 Surface tension (mN/m after 60 min) 41.8 38.7

Solubility index was lowered by enzyme treatment, which indicates abetter heat stability of the milk protein. A better emulsion stabilityof the fat is found as the free fat is lowered. The decrease of surfacetension is indicating less tendency of fouling of dairy equipment.

1-22. (canceled)
 23. A method for producing condensed milk or milkpowder comprising treating a milk composition with a phospholipase andproducing condensed milk or milk powder from the phospholipase treatedmilk composition.
 24. The method of claim 23, wherein the phospholipaseis a phospholipase A and/or phospholipase B.
 25. The method of claim 24,wherein the phospholipase is a phospholipase A used in a concentrationof 0.003-0.3 mg enzyme protein per g milk fat.
 26. The method of claim24, wherein the phospholipase is a phospholipase B used in aconcentration of 0.005-0.5 mg enzyme protein per g milk fat.
 27. Themethod of claim 23, wherein the phospholipase enzyme activity isprovided by an enzyme having essentially only phospholipase activity andwherein the phospholipase enzyme activity is not a side activity. 28.The method of claim 23, wherein the phospholipase activity is providedby an enzyme having another activity as well, e.g. a lipase withphospholipase side activity.
 29. The method of claim 23, wherein thephospholipase is purified.
 30. The method of claim 23, wherein thelecithin content of the milk composition is reduced by at least 5% as aresult of the phospholipase treatment.
 31. The method of claim 23,wherein condensed milk is produced from milk that is standardised to thedesired fat content and subjected to heat treatment.
 32. The method ofclaim 23, wherein condensed milk is produced from milk that is subjectedto heat treatment at 100-120° C. for 1-3 minutes.
 33. The method ofclaim 23, wherein the phospholipase treated milk composition issubjected to evaporation to produce condensed milk.
 34. The method ofclaim 33, wherein the condensed milk is filled into cans or any othersuitable containers and subjected to sterilisation
 35. The method ofclaim 34, wherein the condensed milk is subjected to sterilization at110-120° C. for 15-20 minutes.
 36. The method of claim 33, wherein thecondensed milk is subjected to UHT treatment before filling ontocontainers.
 37. The method of claim 36, wherein the condensed milk issubjected to UHT treatment at 140° C. for 3 seconds.
 38. The method ofclaim 23, wherein milk powder is produced from heat treated milk. 39.The method of claim 23, wherein the phospholipase treatment reduces thedegree of fouling.
 40. Condensed milk or milk powder obtainable by themethod of claim 23.